Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of late-onset autosomal dominant Parkinson's disease (PD), the most prevalent neurodegenerative movement disorder characterized by the selective loss of midbrain dopaminergic neurons (mDAns) of the substantia pars compacta (SNpc). The low penetrance of LRRK2 mutations, the low disease concordance in relatives, and the lack of substantial neurodegeneration in the LRRK2 mouse models would all suggest that the disease may result from a complex interplay between genetic predisposition and exogenous insults, such as the aging process, inflammation, and toxic exposure. In particular, dysfunctional glia-neuron crosstalk might play critical roles in PD physiopathology. Given the unique role of a healthy dialogue between mDAns and their partners glial cells for neuron development and survival, as well as for the response to injury and aging, our research program aimed to investigate whether and how the LRRK2 G2019S mutation might interfere with the establishment and properties of this dialogue, using both in vitro and in vivo animal models. In the first part of this work, we studied the effects of LRRK2 G2019S mutation in the establishment of astrocyte-mDAn crosstalk during postnatal development. Using primary mesencephalic astrocyte-mDAn cultures from wild type (WT) LRRK2 and transgenic (TG) LRRK2 G2019S mice, coupled to different co-culture models, we identified G2019S astrocytes as key mediators of mDAn growth impairment, as reflected by the poor neuronal development and inhibited acquisition of the mature DA phenotype. Next, we addressed potential changes/abnormal responses of TG-LRRK2 G2019S astrocytes, by recapitulating the interaction with inflammatory (LPS) and/or neurotoxic (MPP+) challenges. We found that G2019S astrocytes displayed a significant up-regulation of proinflammatory cytokines, an exaggerated production of reactive oxygen (ROS) and nitrogen (RNS) species, as well as an impairment of mitochondrial dynamics, suggesting that LRRK2 G2019S dysfunctional astrocytes disrupts astrocyte-neuron crosstalk resulting in increased vulnerability and loss of mesencephalic neurons. Significantly, in vivo neonatal exposure of TG-LRRK2 G2019S mice to a subthreshold dose of LPS during the “critical developmental period” for nigrostriatal neurons, sharply increased astrogliosis and significantly reduced TH+ neuron survival in adult life. By contrast in WT mice, neither astrocytes nor TH+ neurons were affected, suggesting that an improper astrocyte-neuron crosstalk during development of LRRK2-G2019S mice might impact on mDAn viability and response to inflammatory triggers in adult life, increasing SNpc neuron vulnerability and cell death. Based on these findings we further investigated the interaction between LRRK2 G2019S and inflammation during aging process, the most critical temporal window for PD, hypothesizing that a chronic low-grade systemic inflammatory stimulus might synergize with LRRK2 G2019S as the mice age to alter the crosstalk between the peripheral and central immune system and to promote nigrostriatal degeneration. To this end four groups of WT and TG LRRK2-G2019S male mice were chronically treated with a sub-threshold dose of LPS at two temporal ages: young adult and middle-aged, and mice were sacrificed at different time intervals during the aging process. We show that low-grade inflammation starting by middle-age promoted mDAn degeneration, which was accompanied by astrogliosis as well as macrophage/monocyte CNS infiltration, being markedly greater in LRRK2 G2019S as compared to WT counterparts. Importantly, brain inflammation, macrophage/monocyte CNS infiltration and mDAn degeneration were preceded by a robust activation of peripheral inflammation, indicating that LRRK2 G2019S synergizes with aging and low-grade inflammation to trigger nigrostriatal dopaminergic degeneration. This new preclinical model will enable the identification of key interactors playing roles in mDAn degeneration, both at central and peripheral levels, and will be useful to explore both the early and age-dependent pathological events for the development of disease-modifying therapies in PD.
Mutazioni del gene Leucine rich repeat kinase 2 (LRRK2) rappresentano le più comuni mutazioni nelle forme ereditarie di morbo di Parkinson (MP), una tra le più comuni malattie neurodegenerative, caratterizzata dalla morte progressiva dei neuroni dopaminergici mesencefalici (mDAn) della substantia nigra pars compacta (SNpc). La bassa penetranza delle mutazioni di LRRK2, la bassa incidenza della malattia nei parenti, unite all’assenza di neurodegenerazione a carico dei neuroni dopaminergici nei modelli murini di MP-LRRK2, suggeriscono che altri fattori genetici e/o ambientali, quali l’infiammazione ed il processo di invecchiamento, possano contribuire all’insorgenza e progressione della malattia. In particolare, una marcata alterazione del dialogo neuroni-glia potrebbe rappresentare un fattore determinante nella progressiva disfunzione e morte neuronale osservati nella MP. In questo progetto di ricerca, usando animali transgenici (TG) portatori della mutazione umana LRRK2 G2019S (TG-G2019S) e animali “wild type”, WT-LRRK2, applicando tecniche di coltura cellulare “in vitro” unite a modelli animali “in vivo”, abbiamo studiato il ruolo della mutazione umana LRRK2 G2019S durante lo sviluppo postnatale delle interazioni astrocita-neurone e durante il processo di invecchiamento. Utilizzando colture primarie derivate dal mesencefalo di topi WT-LRRK2 e TG-G2019S, abbiamo identificato un ruolo inibitorio chiave degli astrociti LRRK2 G2019S nella crescita e differenziamento dei neuroni dopaminergici tirosina-idrossilasi-positivi (TH+). Lo studio della risposta astrocitaria a stimoli infiammatori/ossidativi (LPS, MPP+), hanno rivelato una iper-reattività degli astrociti TG-G2019S, quando paragonati ad astrociti WT, come mostrato dal marcato aumento del rilascio delle maggiori citochine pro-infiammatorie, specie reattive dell’ossigeno (ROS) e dell’azoto (RNS), unite ad una disfunzione mitocondriale significativa. Infine, la somministrazione neonatale di una dose molto bassa di LPS durante una finestra temporale critica per lo sviluppo dei neuroni nigrostriatali determinava nei topi TG LRRK2-G2019S una marcata astrogliosi nella SNpc unita ad una riduzione significativa dei neuroni TH+ in età adulta, mentre non si osservavano differenze del compartimento sia astrogliale, che neuronale nella SNpc dei topi WT trattati con LPS. Questi dati implicano un ruolo importante di LRRK2 G2019S durante lo sviluppo dei neuroni dopaminergici, suggerendo che una disfunzione del compartimento astrocitario possa causare un aumento della vulnerabilità neuronale responsabile di una ridotta sopravvivenza ed aumento della morte neuronale in età adulta. Nella seconda parte di questa tesi, abbiamo studiato l'interazione tra la mutazione LRRK2-G2019S e l'infiammazione durante l'invecchiamento. A tal fine, topi WT-LRRK2 e TG-G2019S giovani adulti (3 mesi) e di mezza età (7 mesi) sono stati trattati cronicamente con una dose molto ridotta di LPS per 12 settimane, e sacrificati a diversi intervalli di tempo durante il processo di invecchiamento. Nei topi TG-G2019S lo stimolo infiammatorio durante la mezza età determinava una marcata degenerazione dei neuroni TH+, quando paragonata alla degenerazione dei neuroni TH+ osservata nei topi WT-LRRK2. Nei topi TG-G2019S, la morte neuronale si accompagnava ad una marcata astrogliosi ed infiltrazione di macrofagi/monociti periferici nel SNC significativamente superiori rispetto ai topi WT. Di maggiore importanza, la morte dei neuroni TH+ nella SNpc, la marcata astrogliosi e l’infiltrazione di macrofagi e monociti periferici nel SNC erano preceduti dalla significativa attivazione del compartimento immunitario periferico. Nell’insieme i dati implicano un’importante sinergia tra mutazione G2019S, invecchiamento ed infiammazione. Questo nuovo modello preclinico consentirà l'identificazione di fattori chiave nel processo neurodegenerativo dei neuroni dopaminergici, sia a livello centrale che periferico, e sarà utile per identificare eventi patologici precoci ed età-dipendenti della MP, con una rilevanza clinica per lo sviluppo di terapie per la MP.
Mutazione LRRK2-G2019S all'interfaccia dell'interazione astrocita- neurone durante lo sviluppo post natale e l'invecchiamento / Giachino, Carmela. - (2022 Nov 23).
Mutazione LRRK2-G2019S all'interfaccia dell'interazione astrocita- neurone durante lo sviluppo post natale e l'invecchiamento
GIACHINO, CARMELA
2022-11-23
Abstract
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of late-onset autosomal dominant Parkinson's disease (PD), the most prevalent neurodegenerative movement disorder characterized by the selective loss of midbrain dopaminergic neurons (mDAns) of the substantia pars compacta (SNpc). The low penetrance of LRRK2 mutations, the low disease concordance in relatives, and the lack of substantial neurodegeneration in the LRRK2 mouse models would all suggest that the disease may result from a complex interplay between genetic predisposition and exogenous insults, such as the aging process, inflammation, and toxic exposure. In particular, dysfunctional glia-neuron crosstalk might play critical roles in PD physiopathology. Given the unique role of a healthy dialogue between mDAns and their partners glial cells for neuron development and survival, as well as for the response to injury and aging, our research program aimed to investigate whether and how the LRRK2 G2019S mutation might interfere with the establishment and properties of this dialogue, using both in vitro and in vivo animal models. In the first part of this work, we studied the effects of LRRK2 G2019S mutation in the establishment of astrocyte-mDAn crosstalk during postnatal development. Using primary mesencephalic astrocyte-mDAn cultures from wild type (WT) LRRK2 and transgenic (TG) LRRK2 G2019S mice, coupled to different co-culture models, we identified G2019S astrocytes as key mediators of mDAn growth impairment, as reflected by the poor neuronal development and inhibited acquisition of the mature DA phenotype. Next, we addressed potential changes/abnormal responses of TG-LRRK2 G2019S astrocytes, by recapitulating the interaction with inflammatory (LPS) and/or neurotoxic (MPP+) challenges. We found that G2019S astrocytes displayed a significant up-regulation of proinflammatory cytokines, an exaggerated production of reactive oxygen (ROS) and nitrogen (RNS) species, as well as an impairment of mitochondrial dynamics, suggesting that LRRK2 G2019S dysfunctional astrocytes disrupts astrocyte-neuron crosstalk resulting in increased vulnerability and loss of mesencephalic neurons. Significantly, in vivo neonatal exposure of TG-LRRK2 G2019S mice to a subthreshold dose of LPS during the “critical developmental period” for nigrostriatal neurons, sharply increased astrogliosis and significantly reduced TH+ neuron survival in adult life. By contrast in WT mice, neither astrocytes nor TH+ neurons were affected, suggesting that an improper astrocyte-neuron crosstalk during development of LRRK2-G2019S mice might impact on mDAn viability and response to inflammatory triggers in adult life, increasing SNpc neuron vulnerability and cell death. Based on these findings we further investigated the interaction between LRRK2 G2019S and inflammation during aging process, the most critical temporal window for PD, hypothesizing that a chronic low-grade systemic inflammatory stimulus might synergize with LRRK2 G2019S as the mice age to alter the crosstalk between the peripheral and central immune system and to promote nigrostriatal degeneration. To this end four groups of WT and TG LRRK2-G2019S male mice were chronically treated with a sub-threshold dose of LPS at two temporal ages: young adult and middle-aged, and mice were sacrificed at different time intervals during the aging process. We show that low-grade inflammation starting by middle-age promoted mDAn degeneration, which was accompanied by astrogliosis as well as macrophage/monocyte CNS infiltration, being markedly greater in LRRK2 G2019S as compared to WT counterparts. Importantly, brain inflammation, macrophage/monocyte CNS infiltration and mDAn degeneration were preceded by a robust activation of peripheral inflammation, indicating that LRRK2 G2019S synergizes with aging and low-grade inflammation to trigger nigrostriatal dopaminergic degeneration. This new preclinical model will enable the identification of key interactors playing roles in mDAn degeneration, both at central and peripheral levels, and will be useful to explore both the early and age-dependent pathological events for the development of disease-modifying therapies in PD.File | Dimensione | Formato | |
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